Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3530-3535, April 1996 Developmental Biology

Twilight-zone and canopy shade induction of the Athb-2 homeobox gene in green plants MONICA CARABELLI*, GIORGIO MORELLIt, GARRY WHITELAMt, AND IDA RUBERTI*§ *Centro di studio per gli Acidi Nucleici, c/o Dipartimento di Genetica e Biologia Molecolare, Universita di Roma La Sapienza P.le Aldo Moro 5, 00185 Rome, Italy; tUnita di Nutrizione Sperimentale, Istituto Nazionale della Nutrizione, Via Ardeatina 546, 00178 Rome, Italy; and TDepartment of Botany, University of Leicester, Leicester LEI 7RH, United Kingdom Communicated by Walter J. Gehring, University of Basel, Basel, Switzerland, December 28, 1995 (received for review October 20, 1995)

ABSTRACT We present evidence that a novel phyto- tomorphogenesis) to (6-14). By contrast, chrome (other than A and B, PHYA and PHYB) in mature green plants, only the phyB mutants show a pheno- operative in green plants regulates the "twilight-inducible" type manifested in changes in shade avoidance, neighbor expression of a plant homeobox gene (Athb-2). Light regula- detection, and flowering (7, 15-17). Thus, PHYB has been tion of theAthb-2 gene is unique in that it is not induced by red proposed to be the major sensor of low R/FR light contrib- (R)-rich daylight or by the light-dark transition but is instead uting to these above developmental processes. However, there induced by changes in the ratio of R to far-red (FR) light. is evidence that PHYB is not solely responsible for these These changes, which normally occur at dawn and dusk responses (16, 18, 19), but as there are no mutants in the other (end-of-day FR), also occur during the daytime under the phytochromes (PHYC, D, and E), it is impossible to test their canopy (shade avoidance). By using pure light sources and respective roles in these and other aspects of plant develop- phyA/phyB null mutants, we demonstrated that the induction ment. of Athb-2 by changes in the R/FR ratio is mediated for the In the present report, we show that changes in light quality most part by a novel operative in green plants. (R/FR ratio), which naturally occur at dawn and dusk (end- Furthermore, PHYB plays a negative role in repressing the of-day FR, EOD-FR) and during the daytime under the accumulation ofAthb-2 mRNA in the dark and a minor role in canopy (shade avoidance), specifically induce the expression of the FR response. The strict correlation of Athb-2 expression a plant homeobox gene, Athb-2 (or HAT4) (20, 21). Moreover, with FR-induced growth phenomena suggests a role for the we demonstrated that the induction ofAthb-2 by changes in the Athb-2 gene in mediating cell elongation. This interpretation R/FR ratio is mediated for the most part by a novel phyto- is supported by the finding that the Athb-2 gene is expressed chrome (other than PHYA and PHYB) operative in green at high levels in rapidly elongating etiolated seedlings. Fur- plants. thermore, as either R or FR light inhibits cell elongation in The unique ability of this plant homeobox gene to respond etiolated tissues, they also down-regulate the expression of to changes in light quality suggests a mechanism by which Athb-2 mRNA. Thus, these data support the notion that plants may adapt their developmental programs in response to changes in light quality perceived by a novel phytochrome changes in the light environment. regulate plant development through the action of the Athb-2 homeobox gene. MATERIALS AND METHODS The sessile nature of plants necessitates the ability to sense and Plant Material and Growth Conditions. The Landsberg respond to changes in environmental stimuli such as light. In erecta ecotype of L. was the wild type fact, changes in light intensity, quality, or duration affect used in this study; the phyA (phyA-1, ref. 12), phyB (hy3-Bo64, important developmental programs throughout the life cycle ref. 6), andphyA phyB (phyA-2 hy3-Bo64, ref. 13) phytochrome of the plant. The initial event of light perception is carried out mutants were used. Plants were grown in a 16-hr light/8-hr by at least three families of photoreceptors that detect differ- dark cycle for 14 days under a high R/FR ratio (R/FR = 20) ent wavelengths within the visible spectrum: phytochrome as described (22). For experiments with etiolated seedlings, [which detects red (R) and far-red (FR) light], a blue light seeds were plated on MS agar plates and stored in darkness for photoreceptor, and an ultraviolet light photoreceptor. Light 2 days at 4°C; germination was induced by placing the plated signal perception by these receptors activates signaling path- seeds in white light for 2 hr at 21°C and growth proceeded in ways leading to the changes in gene expression that underlie darkness at 21°C for 4.5 days. the physiological and developmental responses important Light Sources. The low R/FR (R/FR = 0.93) ratio treat- throughout the life history of a plant (for review, see ref. 1). ment was performed as described (22). For light-pulse exper- To date, the best characterized photoreceptors are the iments, light sources were from Phylips; the bulb type, filters, phytochromes. The photosensory function of these molecules and fluence rates were as follows: FR, bulb Phylinea (60 W), is based on their capacity for reversible interconversion be- filters, FRF 700 (Rohm & Haas) and Roscolux n. 83 (Rosco), tween R- and FR-light-absorbing forms (for review, see refs. and fluence, 350 /W/cm2; R, bulb TL (40W/15), and fluence 1 and 2). InArabidopsis, five genes encoding the phytochrome 316 IXW/cm2. Measurements of fluence rate in the spectral polypeptides, designated PHYA, B, C, D, and E have been regions were performed by using the Photometer IL 150 identified (3, 4). Studies with photomorphogenic mutants (International Light, Newburyport, MA). deficient in functional PHYA and/or PHYB have shed light on RNA Analysis. RNA was isolated and analyzed as described the functions of these two phytochrome species (for review, see (22). To normalize for RNA loading, filters were stripped and ref. 5). In etiolatedArabidopsis plants, both PHYA and PHYB rehybridized with a 3-ATPase (22) probe. Relative amounts of contribute to the switch from dark-growth development (sko- mRNAs were determined by using an Imaging densitometer,

The publication costs of this article were defrayed in part by page charge Abbreviations: FR, far-red; R, red; EOD-FR, end-of-day FR; PHYA, payment. This article must therefore be hereby marked "advertisement" in phytochrome A; PHYB, phytochrome B. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 3530 Downloaded by guest on September 26, 2021 Developmental Biology: Carabelli et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3531 GS-670 (Bio-Rad), and relative transcript levels reported are an average of two experiments.

RESULTS The FR-Light Induction of Athb-2 Gene Expression Is Mediated by a Phytochrome Other than PHYA or PHYB. We A A.hmi have shown (22) that Athb-2 gene expression was induced in LI mature green plants by exposure to 1 hr of FR-rich light. In the .I present report, we investigated whether any phytochrome is ~wil(l involved in this response. Arabidopsis plants were grown for 2 ty,pe weeks in a normal day/night cycle and the steady-state levels I) of Athb-2 mRNA were monitored after different treatments with pure light sources. A 2-min pulse of pure FR light resulted in a 20-fold increase of the Athb-2 mRNA (Fig. 1A, lane 1) compared to controls (Fig. IA, lane 5). A subsequent pulse of R light reversed this effect (Fig. 1A, lane 3), indicating that this response was indeed attributable to the phytochrome system. B By contrast, a pulse of R light had no effect on the Athb-2 .V ^ " I\ transcript levels (Fig. 1A, lane 2). A subsequent pulse of FR i'l ,1 light showed an enrichment of the Athb-2 mRNA similar to that observed with a pulse of FR light alone (Fig. 1A, lane 4). pli.y We next performed parallel experiments in mutant plants lacking functional PHYA and/or PHYB. Surprisingly, inphyA, l) phyB, or phyA phyB mutant plants, a pulse of FR light was able to induce Athb-2 transcript levels to wild-type levels (Fig. 1 B-D, lanes 1) compared to uninduced controls (Fig. 1 B-D, lanes 5). Furthermore, in all phy mutants, Athb-2 FR-light induction was abolished when the FR-light pulse was followed

by a R-light pulse (Fig. I B-D, lanes 3). This FR induction and C Ak.-Ak. R reversibility ofAthb-2 induction observed inphyA, phyB, and *f. *. i'i ".: iA&-.ALI l phyA phyB mutants clearly indicates that a novel phytochrome other than PHYA or PHYB mediates the Athb-2 response to ph.yl FR light. An EOD-FR-Light Treatment Dramatically Affecting De- 1) velopmental Processes Strongly Induces Athb-2 Gene Expres- sion. Time of flowering is developmentally regulated by the length of the day in seed plants. In Arabidopsis, as in other long-day plants, the seedlings show increased hypocotyl elon- gation and an acceleration of flowering in response to FR-light enrichment (e.g., low R/FR ratio). As we showed above that D Athb-2 is artificially induced by a pulse of FR light, we next set 'I out to test whether this gene is induced by an EOD-FR treatment, corresponding to "twilight". Arabidopsis plants were grown in a normal 16-hr day/8-hr night cycle for 2 weeks phvBph]!vll under fluorescent lights (high R/FR). At the end of the light I) period of day 15, plants were exposed to 15 min of FR-rich incandescent light (low R/FR) and transferred to the dark, and RNA was collected at various times after the EOD-FR treat- ment. We performed this analysis on wild-type and phyA 1 2 3 4 5 and/or phyB mutant plants (Fig. 2). FIG. 1. FR-light induction of the Athb-2 mRNA in phyA phyB We found that at the end of the dark period, the Athb-2 mutant plants. Wild-type (A), phyA (B), phyB (C), and phyA phyB (D) transcripts are enriched 15-fold in EOD-FR-light treated plants were grown in 16-hr light/8-hr dark (L/D) cycles for 14 days wild-type plants (Fig. 2A, lane 8, and E) compared to controls under a high R/FR ratio. At day 15, in the middle of the light period, in which no EOD-FR-light treatment was given (Fig. 2A, lane plants were either left in the dark for I hr (control plants, L/D, lane 4, and E). Similar results were obtained with the phyA mutants 5) or exposed to the following light sources and then left in the dark (Fig. 2 B, compare lanes 4 and 8, and E). It is known that for a total of 1 hr. Lanes: 1, 2 min of FR light (L/FR/D); 2, 2 min of wild-type Arabidopsis and phyA mutants respond to an EOD- R light (L/R/D); 3, 2 min of FR light followed by 4 min of R light treatment taller than that did not (L/FR/R/D); 4, 2 min of R light followed by 4 min of FR light FR-light by growing plants (L/R/FR/D). Total RNAs were probed with a 197-bp 3'-end frag- received this treatment (10). The correlation between the ment of Athb-2 cDNA (rows a) and, as a control, with the cDNA EOD-FR induction of the Athb-2 gene and these EOD-FR encoding the ,3 subunit of the mitochondrial ATPase (rows b) ran- phenomena suggests that the Athb-2 gene product may be domly labeled with 32p. inducing these developmental changes. Conversely, mutant phyB seedlings do not display a signif- this interpretation, we found that the Athb-2 mRNA levels are icant elongation in response to an EOD-FR-light treatment constitutively higher both in the light (Fig. 2 C, lane 1, and E) when compared to controls. On the other hand, the phyB and, more significantly, in the dark (Fig. 2 C, lane 4, and E) in mutants elongate more than the wild-type parent under white the phyB mutants compared to wild-type plants (Fig. 2A, lanes or continuous R light (7). The constitutive high elongation of 1 and 4, and E). We also observed that in phyB mutants the these phyB mutants under any light regime suggests that they Athb-2 gene is still inducible by an EOD-FR-light treatment are constitutively derepressed for this process. Consistent with (Fig. 2 C, compare lanes 2 and 6, and E); however, the absolute Downloaded by guest on September 26, 2021 3532 Developmental Biology: Carabelli et al. Proc. Natl. Acad. Sci. USA 93 (1996) L/D EOD FR induction at the end of the night is lower than in wild-type (Fig. 2 C, lane 8, and E). Finally, similar results were also observed 0 1 4 8 0 1 4 8 h in the phyA phyB double mutant (Fig. 2D). Thus, these observations indicate that PHYB and a phytochrome other than PHYA and PHYB are both involved in the Athb-2 A a response to an EOD-FR-light treatment. Athb-2 Expression Is Reversibly Regulated by Changes in wild the Light Quality Occurring During the Daytime. Shade type avoidance in plants involves a developmental response to a reduced R/FR ratio resulting in accelerated growth during the 1 daytime. Many plants react within 5-10 min of exposure to reduced R/FR by upward of a 3- or 4-fold acceleration of extension. Conversely, returning the plant to a high R/FR ratio results in an equally rapid deceleration of extension (23). We have shown (22) that Athb-2 is induced by a 1-hr pulse :.Il of FR-rich-light treatment during the daytime. Here we mea- AdLI-AL, .: sured the kinetics of induction and also its B i: FR-rich-light I, ...: reversibility by increasing R light. Arabidopsis plants were grown for 2 weeks in a normal 16-hr day/8-hr night cycle in phyA fluorescent light (high R/FR ratio). In the middle of day 15, plants were shifted to incandescent light (low R/FR ratio) for b 75 min and then shifted back to fluorescent light for 60 min. RNA was extracted from plants collected at intervals noted in Fig. 3. Exposure of wild-type plants to reduced R/FR resulted in a rapid and strong induction of Athb-2 mRNA reaching a maximum of 35-fold induction (Fig. 3 C andA, lanes 5-7). The C ""LL-Ak.. lidLIl-AL ... level ofAthb-2 transcripts increased sharply during the first 30 'Al a min, reached its maximum after 45 min, and then it remained ., ...... essentially constant (Fig. 3 C and A, lanes 1-7). Moreover, phyB returning the plants to a high R/FR ratio resulted in a rapid decrease of the Athb-2 mRNA levels (Fig. 3 C and A, lanes b 8-12). Physiological studies have shown that mutants deficient in PHYB exhibit reduced shade-avoidance responses. These ob- servations suggested that, although PHYB has an important role in these responses, it is unlikely to be the sole responsible phytochrome (16, 18). We monitored Athb-2 mRNA in phyB mutant plants treated as above to determine the role of PHYB D AL.AL..:A6& c V.... '.1 *" 9a in the kinetics of Athb-2 accumulation and reversibility. Ex- posure ofphyB plants to FR-rich light (low R/FR) resulted in phtyA a 10-fold increase in the levels ofAthb-2 mRNA (Fig. 3 C and phyB B, lanes 1-7). This lower fold induction compared to wild-type b reflects in part a higher basal level expression ofAthb-2 inphyB mutant plants. Conversely, returning the plants to a high R/FR ratio resulted in a rapid decrease of the Athb-2 mRNA level (Fig. 3 C and B, lanes 8-12). Similar results were also observed 1 2 3 4 5 6 7 in thephyA phyB double mutant (data not shown). From these data we conclude that a novel phytochrome other than PHYA or PHYB plays a major role in the Athb-2 response to changes in the R/FR ratio. The Athb-2 Gene Is Down-Regulated by FR and R Light in L/D EOD FR Dark-Grown Seedlings. In all of the above experiments per- E II formed on green light-grown plants, FR light induces elonga- Oh lh 4h 8h 0 h I h 4h tion and also induces Athb-2 gene expression. Conversely, in 1.0 1.0 1.3 1.0 1.0 10.0 9.1 15.0 etiolated is inhibited continu- wt _ plants hypocotyl elongation by phyA 1.1 1.0 1.3 1.1 1Fi 1.0 10.6 9.6 19.5 ous exposure to either R or FR light (for review, see ref. 1). 1.9 1.9 2.6 5.0 1.8 8.3 8.6 10.0 As Athb-2 mRNA induction FR-rich phyB --t- by light parallels elonga- 1.9 2.5 9.7 10.7 phyAphyB _ 1.8 I . 25 I 3.8I 1.8 I I I tion in light-grown plants, we set out to determine whether the expression of this homeobox gene is affected by light in etiolated FIG. 2. Effect of an EOD-FR-light treatment on theAthb-2 mRN.[A plants. level in wild-type and mutant plants. Wild-type (A), phyA (B), phy,B We monitored Athb-2 mRNA in dark-grown etiolated wild- (C), and phyA phyB (D) plants were grown in 16-hr light/8-hr dairk type seedlings and tested the effects of pulses of pure R or FR (L/D) cycles for 14 days under a high R/FR ratio (R/FR = 20); at daay light. Remarkably, we observed that in etiolated seedlings a 15, the plants were either left in the same day/night cycle (contrl ol plants) or transferred to a single cycle consisting of 16 hr of liglht followed by 15 min of FR-rich light (R/FR = 0.93) followed by 7.775 and 8 hr (lanes 4 and 8) during the night period. Rows: a, Athb-2 hr of darkness. Total RNA was isolated from control plants (L/D) anid mRNA; b, /3-ATPase mRNA. (E) Quantitation of Athb-2 mRNA EOD-FR-light-treated plants (EOD FR) at the end of the day peric>d expressed as the relative transcript level, which refers to the ratio of (0 hr, lanes 1 and 5) and at 1 hr (lanes 2 and 6), 4 hr (lanes 3 and 77), the Athb-2 transcript to the 3-ATPase transcript. Downloaded by guest on September 26, 2021 Developmental Biology: Carabelli et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3533

lo\ R: i'R hiiuh R:FR 5 I15 30 45 60 75 O8 90 105 120 135 miln A wild( (ype

.. A ...... I) to a wild type B ' 64 -- V b +,. i *....

I)

1 2 3 4 5 6 7 9 10 I 1 2 B c too RTI, phyA 10

J &- , * . i b

I ) l wt [; 20 phyB C I1( ca ::::.:. ..'. phyB -:-:-7-7- '-- .1:.:.:. *,-j-- ..I I.:.,>-- r,^------I. 1,--j11^-T , I. I\ 1 I J. 1 5 15 3) 1 70( 5 I S) t)) 105 120 135 min ) FIG. 3. Effect of low and high R/FR ratio on the Athb-2 mRNA level in wild-type and phyB mutant plants. Wild-type (A) and phyB (B) plants were grown in 16-hr light/8-hr dark cycles for 14 days under a high R/FR ratio (R/FR = 20). At day 15, total RNA was prepared either from plants collected in the middle of the day period (control plants, 0 min, lane 1) or from plants exposed to a low R/FR ratio (R/FR = 0.93) for 5 min (lane 2), 15 min (lane 3), 30 min (lane 4), 45 D min (lane 5), 60 min (lane 6), and 75 min (lane 7) or from plants exposed to a low R/FR ratio for 75 min and then returned to a high plivA R/FR ratio (R/FR = 20) for 5 min (lane 8), 15 min (lane 9), 30 min phvA (lane 10), 45 min (lane 11), and 60 min (lane 12). Numbers above the /,h:,B lanes refer to the time from the beginning of the exposure to low R/FR 1b light. Rows: a,Athb-2 mRNA; b, ,3-ATPase mRNA. (C) Quantitation of Athb-2 mRNA expressed as the relative transcript level (RTL). pulse of either FR or R light resulted in a 4-fold reduction of 1 2 3 4 5 Athb-2 mRNA (Fig. 4A, lanes 2 and 3) compared to control lane As the of Athb-2 in FIG. 4. Effect of FR- and R-light pulses on theAthb-2 mRNA level seedlings (Fig. 4, 1). down-regulation in etiolated wild-type and mutant seedlings. Wild-type (A), phyA (B), etiolated seedlings occurs with both pulses of R and FR light, phyB (C), and phyA phyB (D) seedlings were grown for 4.5 days in the this result would appear to suggest that this repression is the dark and then either left in the dark (control seedlings, D/D, lane 1) result of a nonreversible phytochrome-mediated photore- or exposed to the following light sources and then returned to the dark sponse. Similar phenomena (described as very-low-fluence for a total of 1 hr. Lanes: 2, 2 min of FR light (D/FR/D); 3, 2 min of responses that can be triggered by both R and FR light) have R light (D/R/D); 4, 2 min of FR light followed by 4 min of R light been attributed to a very low level of active PHYA needed for (D/FR/R/D); 5, 2 min of R light followed by 4 min of FR light these photoresponses (24). If this was the case, we would (D/R/FR/D). Rows: a, Athb-2 mRNA; b, /3-ATPase mRNA. expect both responses to be absent in a phyA null mutant. Surprisingly, we found that only the FR-induced inhibition of and 5). By contrast, FR reversibility of R-light repression can Athb-2 is lost in the phyA mutant (Fig. 4B, lane 2). By contrast, be observed in both phyA and phyA phyB etiolated seedlings the Athb-2 gene is down-regulated by R and FR light in phyB (Fig. 4 B and D, lane 5). This indicates that the R repression mutants, at levels similar to wild type (Fig. 4C, lanes 2 and 3). and the FR reversion of this response is indeed attributable to Moreover, the phyA phyB double mutant responds like the a phytochrome system other than PHYA or PHYB. phyA single mutant (Fig. 4D, lanes 2 and 3). To prove that the above light responses are indeed mediated DISCUSSION by a phytochrome, we examined the R/FR reversibility. In the case of wild type, as each FR and R light represses Athb-2, We report the first example, to our knowledge, of a gene there is no expected or observed reversibility (Fig. 4A, lanes 4 regulated by light quality and a phytochrome operative in Downloaded by guest on September 26, 2021 3534 Developmental Biology: Carabelli et al. Proc. Natl. Acad. Sci. USA 93 (1996)

green plants. The light regulation of the Athb-2 gene is unique is derepressed inphyB mutants in both the daytime and during in that it is not induced by R-rich daylight or by the light/dark the night. transition but is instead induced by changes in the R/FR ratio. Finally, the demonstration that Athb-2 gene expression is A high R/FR ratio represses Athb-2 gene expression, while regulated by light in the phyA phyB double mutant has FR-rich light (low R/FR) induces the expression of this uncovered, to our knowledge, for the first time the existence homeobox gene in green plants. Furthermore, we showed that of a novel functional phytochrome in green plants that might the expression of the Athb-2 gene is regulated by a novel be responsible, together with PHYB, for the activation of phytochrome other than PHYA or PHYB, which works in physiological responses in FR-enriched light (low R/FR ratio). combination with PHYB in green plants in both daytime and All the results in green plants suggest a possible link between nighttime. the light-induced expression of Athb-2 and the light-induced In the past, the effects of phytochrome on gene expression elongation processes. In support of the hypothesis that the were studied primarily in etiolated plants, where the most Athb-2 homeobox gene might have a role in cell elongation, we dramatic responses to pulses of R light were observed. Several showed that Athb-2 gene expression is high in elongated genes, many of which are involved in (Cab, etiolated seedlings and is repressed by either R or FR light. RbcS), were found to be induced to high levels upon exposure Our initial discovery thatAthb-2 is repressed by either R or FR of etiolated seedlings to R light (for review, see refs. 25 and 26). light in etiolated plants suggested that this effect was the result By contrast, other genes, such as those encoding NADPH- of a very-low-fluence response mediated by a single phyto- protochlorophyllide oxidoreductase (27), PHYA (28), aspar- chrome (PHYA). However, phyA and phyB mutant analysis agine synthetase (29), and other unidentified genes (30), were clearly indicates that the R and FR effects are instead medi- found to be repressed upon exposure to light also in etiolated ated via distinct phytochromes. This finding for Athb-2 gene plants. regulation suggests that some of the very-low-fluence re- Despite the importance of light on the regulation of plant sponses induced by either R- or FR-light pulses may also be the development, at present there is little information on gene result of distinct phytochromes operating in etiolated plants. responses to phytochrome in mature green plants. Many Thus, these very-low-fluence responses should be reexamined light-induced mRNAs decline when green plants are placed in in the available phy mutant backgrounds. Our studies of phyA the dark for extended periods and increase again upon sub- and/or phyB mutant plants also indicate that a novel phyto- sequent illumination. The fact that normal levels of mRNA for chrome (other than PHYA and PH-YB) is responsible, together several light-responsive genes was found in light-grown leaves with PHYA, for the down-regulation of Athb-2 in etiolated of several Arabidopsis mutants with low or undetectable levels seedlings. This novel phytochrome may be the same or a of spectrally active phytochrome has been interpreted to mean distinct phytochrome from the one which regulates Athb-2 that phytochrome plays only a minor role in gene regulation in gene expression in green plants. green tissues (31). The Athb-2 gene may now be used in the mutant screening The lack-of a significant phytochrome-regulated gene ex- strategies to isolate mutations in this novel phytochrome(s) pression in green plants was incongruous with the physiolog- affecting developmental responses in green and etiolated ical data in living plants where changes in the R/FR ratio plants. Further studies on the Athb-2 homeobox gene should dramatically affect plant development. However, most of the elucidate how it elicits these light-induced changes in the light-regulated genes studied thus far have been structural developmental programs/pathways. genes involved in photosynthesis. Here we report that a putative developmental regulatory gene, Athb-2, is indeed We are grateful to G. Coruzzi for helpful discussion and critical regulated by changes in light quality that naturally occur daily reading of this manuscript. We thank R. Gargamelli for photographic during the life of a plant. The regulation of this gene by changes assistance. This research was supported partly by the Fondazione in light quality (the R/FR ratio) is coincident with documented Istituto Pasteur-Fondazione Cenci Bolognetti, Universita di Roma La effects of light on important aspects of plant growth and Sapienza and by National Research Council of Italy, Special Project is a of the For in the ratio occur Raisa, Sub-project N.2, Paper number 2517. M.C. fellow development. example, changes R/FR National Research Council of at the beginning of the day (dawn) and at the end of the day Italy. in the of other and in (dusk), proximity vegetation canopy 1. Quail, P. H. (1994) Curr. Opin. Genet. Dev. 4, 652-661. shade (32). The latter two situations induce the so-called 2. Furuya, M. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, shade-avoidance responses that produce dramatic changes in 617-645. the development of a plant. In fact, these situations result in a 3. Sharrock, R. A. & Quail, P. H. (1989) Genes Dev. 3, 1745-1757. redirection of resources and growth potential from leaf and 4. Clack, T., Mathews, S. & Sharrock, R. A. (1994) Plant Mol. Biol. storage organs into increased extension growth of internode 25, 413-428. and petioles in a plant effort to optimize light capture (23). 5. Whitelam, G. C. & Harberd, N. P. (1994) Plant Cell Environ. 17, The unique ability of Athb-2 to respond to changes in light 615-625. that occur the 6. Koornneef, M., Rolff, E. & Spruit, C. J. P. (1980) Heynh. Z. quality (R/FR ratio) normally during twilight 147-160. zones and and in the shade reinforces our Pflanzenphysiol. 100, (dawn dusk) original 7. Nagatani, A., Chory, J. & Furuya, M. (1991) Plant Cell Physiol. hypothesis that this homeobox gene regulates photomorpho- 32, 1119-1122. genic phenomena such as neighbor detection and shade avoid- 8. Somers, D. E., Sharrock, R. A., Tepperman, J. M. & Quail, P. H. ance (22). In Arabidopsis, PHYB appears to play an important (1991) Plant Cell 3, 1263-1274. role in the regulation of these processes, because phyB null 9. Nagatani, A., Reed, J. W. & Chory, J. (1993) Plant Physiol. 102, mutants do not respond to the EOD-FR treatments and do not 269-277. show most of the shade-avoidance reactions (7, 16). The 10. Parks, B. M. & Quail, P. H. (1993) Plant Cell 3, 1177-1186. absence of PHYB in these mutants leads to excessive elonga- 11. Reed, J. W., Nagpal, P., Poole, D. S., Furuya, M. & Chory, J. tion of stem, and indicating that PHYB acts (1993) Plant Cell 5, 147-157. hypocotyl, petiole, 12. G. as a of cell In addition, mutants Whitelam, C., Johnson, E., Peng, J., Carol, P., Anderson, repressor elongation. phyB M. L., J. S. & N. P. Plant Cell 5, 757-768. an of for and Cowl, Harberd, (1993) have absolute requirement light germination (33) 13. Johnson, E., Bradley, M., Harberd, N. P. & Whitelam, G. C. show an accelerated rate of flowering (15, 16). Similar phe- (1994) Plant Physiol. 105, 141-149. notypes have been observed in Athb-2-overexpressing Arabi- 14. Reed, J. W., Nagatani, A., Elich, T. D., Fagan, M. & Chory, J. dopsis plants (34), suggesting a direct involvement ofAthb-2 in (1994) Plant Physiol. 104, 1139-1149. light-mediated growth phenomena. This hypothesis is sup- 15. Goto, N., Kumagai, T. & Koornneef, M. (1991) Physiol. Plant 83, ported by the data reported herein in which the Athb-2 mRNA 209-215. Downloaded by guest on September 26, 2021 Developmental Biology: Carabelli et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3535 16. Whitelam, G. C. & Smith, H. (1991) J. Plant Physiol. 139, 25. Gilmartin, P. M., Sarokin, L., Memelink, J. & Chua, N.-H. (1990) 119-125. Plant Cell 2, 369-378. 17. Chory, J. (1992) Development (Cambridge, U.K) 115, 337-354. 26. Thompson, W. F. & White, M. J. (1991)Annu. Rev. Plant Physiol. 18. Robson, P. R. H., Whitelam, G. C. & Smith, H. (1993) Plant Plant Mol. Biol. 42, 423-466. 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